Apoptosis, or programmed cell death, is a biochemical pathway critical to normal development and tissue homeostasis in multi-cellular organisms. Aberrant suppression of apoptosis is a requisite contributor to human cancer. The long-term objective of this application is to elucidate the intracellular pathways and the molecular mechanisms that lead to apoptosis. The Bcl-2 family proteins are major regulators of apoptosis. Genetic and biochemical studies establish that two of the Bcl-2 family proteins, Bax and Bak, are requisite mediators of the mitochondria-dependent apoptotic pathway. In order to initiate apoptosis, Bax needs to undergo an activation process, which involves its mitochondrial translocation, the formation of Bax homo- oligomers, and the subsequent damage of mitochondria, a hallmark of apoptosis. The mechanisms of this activation process remain poorly understood. Using techniques of biochemistry and molecular biology, this proposal aims at determining the molecular mechanisms of Bax activation during apoptosis. In Aim 1, we will determine the mechanism of mitochondrial translocation of Bax. The role of mitochondrial targeting sequence(s), conformational changes, and dimeric states of Bax in its mitochondrial translocation will be examined. In Aim 2, the helices and amino acid residues critical for the oligomerization of Bax will be defined. Mutational study and biochemical assays will be used to examine the role of homo-oliomerization in the apoptotic activity of Bax. In Aim 3, we will determine the mechanism of Bax activation by upstream activators. In addition, loss-of-functionstudies using siRNA will be combined with in vitro studies to examine the role of other Bcl-2 family proteins in the activation of Bax during apoptosis. These studies will determine the molecular mechanisms of Bax activation and will help elucidate the pathways that lead to apoptosis. Upon a successful completion, this proposal will not only provide basic knowledge on the regulation of apoptosis, but may also provide new insights on the design of therapeutic agents against cancer.